Bottom Line:
Dense deposit disease (DDD) is strongly associated with the uncontrolled activation of the complement alternative pathway.Thus, the reduction in GBM C3 was dependent on the ability of mCFH to regulate C3 activation in plasma.The implication is that successful therapy of DDD is likely to be achieved by therapies that inhibit C3 turnover in plasma.

ABSTRACTDense deposit disease (DDD) is strongly associated with the uncontrolled activation of the complement alternative pathway. Factor H (CFH)-deficient (Cfh(-/-)) mice spontaneously develop C3 deposition along the glomerular basement membrane (GBM) with subsequent development of glomerulonephritis with features of DDD, a lesion dependent on C3 activation. In order to understand the role of CFH in preventing renal damage associated with the dysregulation of the alternative pathway we administered purified mouse CFH (mCFH) to Cfh(-/-) mice. 24h following the administration of mCFH we observed an increase in plasma C3 levels with presence of intact C3 in circulation showing that mCFH restored control of C3 activation in fluid phase. mCFH resulted in the reduction of iC3b deposition along the GBM. The exogenous mCFH was readily detectable in plasma but critically not in association with C3 along the GBM. Thus, the reduction in GBM C3 was dependent on the ability of mCFH to regulate C3 activation in plasma. Western blot analysis of glomeruli from Cfh(-/-) mice demonstrated the presence of iC3b. Our data show that the C3 along the GBM in Cfh(-/-) mice is the C3 fragment iC3b and that this is derived from plasma C3 activation. The implication is that successful therapy of DDD is likely to be achieved by therapies that inhibit C3 turnover in plasma.

fig1: Plasma C3 levels and state in Cfh−/− mice injected with purified mCFH. (A) Plasma C3 levels in Cfh−/− mice 24 h after the injection of PBS, 1 mg of purified mouse factor H or 0.75 μg of LPS. Wild-type C3 control level in this ELISA was 387 μg/ml. Columns denote median values with standard deviation (B) Western blot for mouse C3 using EDTA plasma from Cfh−/− mice 24 h after the injection of PBS, mCFH (1 mg) or LPS (0.75 μg) under reducing conditions. The EDTA plasma dilution used for all the samples was 1 in 100. LPS: lipopolysaccharide, PBS: phosphate-buffered saline.

Mentions:
To investigate whether the administration of purified mCFH could restore control of AP activation in plasma we assessed the level and state of circulating C3 in Cfh−/− mice after mCFH administration. Plasma C3 levels in unmanipulated Cfh−/− mice are markedly reduced with median levels of approximately 5% of wild-type levels (Pickering et al., 2002). Administration of 1 mg of our purified mCFH to Cfh−/− mice resulted in an increase in plasma C3 levels at 24 h (Fig. 1a). Since our mCFH preparation contained LPS we also assessed plasma C3 levels in three Cfh−/− mice that received 0.75 μg (twice the amount of LPS detected in the administered mCFH preparation) of purified LPS alone. At 24 h these mice had an increase in plasma C3 levels similar to Cfh−/− mice that had received mCFH (Fig. 1a). We next assessed the activation state of the plasma C3 using western blotting under reducing conditions (Fig. 1b). This allowed identification of C3 α-chain fragments thereby enabling us to discriminate between intact C3 and its proteolytic fragments (C3b, iC3b and C3dg). Intact α-chain was only detectable in the Cfh−/− mice that had received mCFH (Fig. 1b, far right lane). In contrast, in Cfh−/− mice injected with LPS or PBS no intact C3 α-chain was present. In these animals, the C3 β-chain was present together with C3 α-chain fragments consistent with ongoing plasma C3 activation. Taken together, this data shows that whilst either LPS or mCFH can increase total antigenic C3 levels in plasma, only mCFH was able to regulate AP activation allowing intact plasma C3 to circulate in the Cfh−/− mice.

fig1: Plasma C3 levels and state in Cfh−/− mice injected with purified mCFH. (A) Plasma C3 levels in Cfh−/− mice 24 h after the injection of PBS, 1 mg of purified mouse factor H or 0.75 μg of LPS. Wild-type C3 control level in this ELISA was 387 μg/ml. Columns denote median values with standard deviation (B) Western blot for mouse C3 using EDTA plasma from Cfh−/− mice 24 h after the injection of PBS, mCFH (1 mg) or LPS (0.75 μg) under reducing conditions. The EDTA plasma dilution used for all the samples was 1 in 100. LPS: lipopolysaccharide, PBS: phosphate-buffered saline.

Mentions:
To investigate whether the administration of purified mCFH could restore control of AP activation in plasma we assessed the level and state of circulating C3 in Cfh−/− mice after mCFH administration. Plasma C3 levels in unmanipulated Cfh−/− mice are markedly reduced with median levels of approximately 5% of wild-type levels (Pickering et al., 2002). Administration of 1 mg of our purified mCFH to Cfh−/− mice resulted in an increase in plasma C3 levels at 24 h (Fig. 1a). Since our mCFH preparation contained LPS we also assessed plasma C3 levels in three Cfh−/− mice that received 0.75 μg (twice the amount of LPS detected in the administered mCFH preparation) of purified LPS alone. At 24 h these mice had an increase in plasma C3 levels similar to Cfh−/− mice that had received mCFH (Fig. 1a). We next assessed the activation state of the plasma C3 using western blotting under reducing conditions (Fig. 1b). This allowed identification of C3 α-chain fragments thereby enabling us to discriminate between intact C3 and its proteolytic fragments (C3b, iC3b and C3dg). Intact α-chain was only detectable in the Cfh−/− mice that had received mCFH (Fig. 1b, far right lane). In contrast, in Cfh−/− mice injected with LPS or PBS no intact C3 α-chain was present. In these animals, the C3 β-chain was present together with C3 α-chain fragments consistent with ongoing plasma C3 activation. Taken together, this data shows that whilst either LPS or mCFH can increase total antigenic C3 levels in plasma, only mCFH was able to regulate AP activation allowing intact plasma C3 to circulate in the Cfh−/− mice.

Bottom Line:
Dense deposit disease (DDD) is strongly associated with the uncontrolled activation of the complement alternative pathway.Thus, the reduction in GBM C3 was dependent on the ability of mCFH to regulate C3 activation in plasma.The implication is that successful therapy of DDD is likely to be achieved by therapies that inhibit C3 turnover in plasma.

ABSTRACTDense deposit disease (DDD) is strongly associated with the uncontrolled activation of the complement alternative pathway. Factor H (CFH)-deficient (Cfh(-/-)) mice spontaneously develop C3 deposition along the glomerular basement membrane (GBM) with subsequent development of glomerulonephritis with features of DDD, a lesion dependent on C3 activation. In order to understand the role of CFH in preventing renal damage associated with the dysregulation of the alternative pathway we administered purified mouse CFH (mCFH) to Cfh(-/-) mice. 24h following the administration of mCFH we observed an increase in plasma C3 levels with presence of intact C3 in circulation showing that mCFH restored control of C3 activation in fluid phase. mCFH resulted in the reduction of iC3b deposition along the GBM. The exogenous mCFH was readily detectable in plasma but critically not in association with C3 along the GBM. Thus, the reduction in GBM C3 was dependent on the ability of mCFH to regulate C3 activation in plasma. Western blot analysis of glomeruli from Cfh(-/-) mice demonstrated the presence of iC3b. Our data show that the C3 along the GBM in Cfh(-/-) mice is the C3 fragment iC3b and that this is derived from plasma C3 activation. The implication is that successful therapy of DDD is likely to be achieved by therapies that inhibit C3 turnover in plasma.